Immobilisation of chelating groups for immobilised metal ion chromatography (imac)

ABSTRACT

The present invention refers to a method for binding a polycarboxylic acid to a solid phase. Further, the invention refers to a solid phase having a polycarboxylic acid immobilized thereto and methods of using the solid phase, e.g. for purifying His-tagged recombinant polypeptides.

This application is a divisional of Ser. No. 12/672,345, filed Feb. 5,2010, which is a 35 U.S.C. 371 National Phase Entry Application fromPCT/EP2008/006485, filed Aug. 6, 2008, which claims the benefit ofEuropean Patent Application Nos. 07015389.5 and 08012686.5 filed on Aug.6, 2007 and Jul. 14, 2008 and which claims the benefit of U.S. Ser. No.60/954,144 filed Aug. 6, 2007, the disclosures of which are incorporatedherein in their entirety by reference.

The present invention refers to a method for binding a polycarboxylicacid to a solid phase. Further, the invention refers to a solid phasehaving a polycarboxylic acid immobilized thereto and methods of usingthe solid phase, e.g. for purifying recombinant polypeptides.

A very powerful strategy for purifying recombinant proteins is the useof a “His-tag”, which comprises typically 6-10 consecutive histidines.His-tags bind tightly to free coordination sites of Ni²⁺-ions. They canbe released by a high concentration of imidazole, which competes forcoordination sites on Ni²⁺. Such cycle of specific absorption anddesorption can be used for one-step purification of a desired protein,resulting in enrichment factors of 100 or even more.

The most widely used variant of this technique usesN_(α)N_(α)-bis(carboxymethyl)-lysine coupled via the ε-amino group toagarose beads. The active group is NTA (nitrilo triacetic acid) chargedwith Ni²⁺, while the spacer is an amino butyl group. SuchNi²⁺-NTA-matrix has, however, several serious disadvantages, such as thehigh costs for N_(α)N_(α)-bis(carboxymethyl)-lysine.

An additional disadvantage is the instability of the immobilisedNi²⁺-ions. Because NTA has only 4 coordination sites for Ni²⁺, the Ni²⁺ions leak easily from the matrix and contaminate the protein samples.This is a severe problem for at least two reasons: Ni²⁺ is a rathertoxic heavy metal and it catalyses undesired oxidation of the proteinsample. Furthermore, NTA-bound Ni²⁺ is easily reduced byprotein-protecting agents such as DTT (dithiothreitol) and then releasedfrom the matrix. Finally, metal-chelating protease inhibitors such asEGTA or EDTA cannot be combined with a Ni²⁺-NTA-matrix, because theyextract the Ni²⁺-ions from this matrix.

U.S. Pat. No. 6,670,159 describes a method for the preparation of metalchelate conjugates based on NTA, wherein NTA or a salt thereof isreacted in an aqueous medium at an alkaline pH of at least 8 with aproteinaceous molecule containing a primary amine group in the presenceof carbodiimide.

WO 2004/036189 discloses a separation method for polypeptides using ametal chelate modified support. This support comprises NTA bound to anamino-modified solid phase via a carboxamide group.

GB 2 067 203 discloses polymeric chelating agents comprising a supportbound thereto a mixture of EDTA amides bound via a single carboxamidegroup or via two carboxamide groups. The use of this support for thepurification of polypeptides is neither disclosed nor suggested.

US 2004/204569 discloses a His-tag protein comprising a (His-Asn)₆-tag.

US 2002/164718 discloses affinity peptides for immobilised metal ionchromatography (IMAC) comprising a spaced His-tag with an amino acidsequence consisting of His, two aliphatic or amide amino acids, His,three basic or acidic amino acids, His, and an aliphatic or amide aminoacid.

The object according to the present invention was to provide novelmethods and compositions suitable for immobilised metal ionchromatography (IMAC) to circumvent the problems associated with theprior art.

Surprisingly it was found that an EDTA-based solid phase complexed withNi²⁺, particularly a solid phase wherein a carboxyl group of EDTA isbound to amino groups on the solid phase, has excellent properties inimmobilized metal ion chromatography (IMAC) applications, particularlyfor the purification of recombinant polypeptides comprisingpoly-histidine tags. This finding was fully unexpected: According to theexisting literature, an EDTA-based solid phase should not be suitablefor nickel chelate chromatography, because this hexadentate chelatorshould occupy all six coordinations on Ni²⁺, supposedly leaving none forthe binding of a histidine residue. In contrast to this expectation, itwas found that an EDTA-based solid phase not only exhibits a stablebinding of transition metal ions, particularly of Ni²⁺ ions, but alsothat the resulting complexes with Ni²⁺, Zn²⁺, Co²⁺ or Cu²⁺ have a highselectivity for histidine-tagged proteins.

A first aspect of the present invention refers to the use of animmobilized chelator having six or more coordination groups forimmobilized metal ion chromatography (IMAC), particularly forpurification of poly-histidine-tagged proteins. Preferably, theimmobilized chelator is a polycarboxylic acid amide or ester having sixor more coordination groups, which are particularly selected from amino,carboxyl, carboxamide and hydroxamate groups.

More preferably, the immobilized chelator is a solid phase having apolycarboxylic acid immobilized thereto having a structure of formula(Ia) or (Ib):

wherein SP is a solid phase;

R¹ is hydrogen or an organic residue that does not interfere with theapplication of the solid phase, e.g. a C₁-C₃ alkyl radical, and

PCA is the residue of a polycarboxylic acid, particularly of an aminopolycarboxylic acid, or a salt thereof.

Even more preferably, the solid phase has a structure of formula (II):

wherein SP is the solid phase; and

one or more of the carboxylic acid groups may be deprotonated.

The solid phase-bound polycarboxylic acid (Ia), (Ib) or (II) may becomplexed with a polyvalent metal ion, e.g. a Ni²⁺ ion.

A further aspect of the present invention refers to a method for bindinga polycarboxylic acid having 6 or more coordination groups to a solidphase, comprising the steps: (a) providing a polycarboxylic acid and asolid phase comprising amino groups, (b) reacting the amino groups withthe polycarboxylic acid in the presence of a condensing agent, whereinthe condensing agent is present in a molar excess over the amino groupsand the polycarboxylic acid is present in molar excess over thecondensing agent and the amino groups, and wherein a single carboxylgroup of the polycarboxylic acid reacts with the amino groups.

Still a further aspect of the present invention refers to a solid phasehaving a polycarboxylic acid immobilized thereto having a structure offormula (Ia) or (Ib):

wherein SP is a solid phase;

R¹ is hydrogen or an organic residue that does not interfere with theapplication of the solid phase, e.g. a C₁-C₃ alkyl radical,

PCA is the residue of a polycarboxylic acid, particularly of an aminopolycarboxylic acid, or a salt thereof,

wherein the immobilized polycarboxylic acid amide or ester has at least6 or more coordination groups, which are particularly selected fromamino, carboxyl, carboxamide and hydroxamate groups,

and wherein the surface of the solid phase is preferably substantiallyfree from accessible amino groups, wherein e.g. ≧90%, preferably ≧95%and more preferably ≧99% of the accessible amino groups on the surfaceof the solid phase are blocked.

In some embodiments of the invention, the carboxamide group —NR¹—CO— isdirectly bound to the solid phase. In other embodiments, the carboxamidegroup is bound to the solid phase via a linker, which may have a lengthof 1 atom up to 20 atoms, preferably 2-12 atoms, e.g. 2-6 atoms whichmay be selected from carbon atoms and optionally heteroatoms such as Oand/or N.

Still a further aspect of the present invention is a method of purifyinga recombinant polypeptide comprising the steps: (a) providing a samplecomprising a polypeptide with a plurality of histidine residues, e.g. aplurality of consecutive histidine residues, (b) contacting the samplewith a solid phase as described above that contains a pre-boundcomplexing metal ion, e.g. a Ni²⁺ ion, under conditions wherein therecombinant polypeptide selectively binds to the solid phase, (c)separating the bound recombinant polypeptide from other samplecomponents, and (d) eluting the recombinant polypeptide from the solidphase.

Still a further aspect of the present invention is a recombinantpolypeptide comprising a “spaced histidine tag” with at least 4histidine residues in a sequence [H_(n)S_(m)]_(k) wherein H ishistidine, S is an amino acid residue different from histidine selectedfrom glycine and/or serine and/or threonine, n is in each caseindependently 1-4, m is in each case independently 1-6, and k is 2-6,preferably 2-5. The spaced histidine tag may have a regular sequence,i.e. n and m have in each occurrence the same value or an irregularsequence, i.e. n and m may have different values. A larger number ofhistidines within a polyhistidine-tag may increase binding strength andspecificity for a Ni²⁺ chelate matrix. Too many consecutive histidines,however, may lower expression levels and solubility of recombinantproteins, e.g. proteins recombinantly expressed in E. coli. Theseproblems can be overcome by interrupting continuous runs of histidineswith short spacers comprising glycine, serine and/or threonine, i.e.with a spaced histidine tag. Recombinant proteins, such as nucleartransport receptors or dihydrofolate reductase (DHFR) show higherexpression and better solubility during expression in Escherichia coli,when tagged with a spaced histidine tag according to the presentinvention as compared to a conventional oligo histidine tag.

According to the present invention a method for binding a polycarboxylicacid to a solid phase is provided. The polycarboxylic acid has 6 ormore, e.g. 6, 7 or 8 coordination groups. Preferably the polycarboxylicacid has 4, 5 or more carboxylic acid groups. The polycarboxylic acidmay be an amino, nitrilo or ether polycarboxylic acid, wherein aminopolycarboxylic acids, particularly amino polycarboxylic acids withtertiary amino groups are preferred. Specific examples of polycarboxylicacids are ethylene diamino tetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),diethylene triamino pentaacetic acid (DTPA) andtriethylenetetramine-N,N,N′,N′,N″,N″ hexaacetic acid (TTHA). Especiallypreferred is EDTA.

The polycarboxylic acid is preferably bound to a solid phase comprisingprimary or secondary amino groups. The binding is carried out underconditions which allow selective binding of only one carboxyl group ofthe polycarboxylic acid to the solid phase without the need of isolatingan activated or derivatized form of the polycarboxylic acid such as ananhydride, an active ester, or a form where a primary or secondary aminegroup of the polycarboxylic acid has been kept accessible for asubsequent coupling step. By means of the reaction between carboxylgroup and amino groups a carboxamide bond is formed. The solid phasemay, e.g. be an amino-functionalized chromatographic support. Forexample, the solid phase may be selected from amino-functionalizedcarbohydrates such as agarose, sepharose, or cellulose, metals orsemi-metals such as silicon or oxides thereof such as silica, glass,plastics such as polystyrene or lipids such as phosphatidylethanolamine-or phosphatidylserine-containing phospholipids. Further, the solid phasemay comprise particles, e.g. vesicles, magnetic beads, quantum dots,vesicles, or proteins. The amino groups on the solid phase arepreferably primary or secondary amino groups, e.g. aliphatic primaryamino groups.

The amino-functionalization of the solid phase may be carried out byknown methods, e.g. by reacting an amino group-containing silane such asamino propyl triethoxysilane with a solid phase such as silica or glassor by reacting ammonia with an epoxy-activated solid phase. Preferably,the amino-functionalization of silica or glass is carried out withaminopropyl trimethoxy silane or aminopropyl triethoxy silane.Carbohydrates, such as agarose, sepharose, or cellulose are preferablyfirst epoxy-activated e.g. with epichlorohydrine or epibromohydrine toyield epoxy-activated matrices and then treated with excess of ammonia,a primary amine, or a hydroxylamine to yield amino-modified matrices.

The amino group density on the solid phase can be varied by the reactionconditions of the amino-functionalization, e.g. temperature, durationand/or concentration of reactants. For example, theamino-functionalizing agent may be diluted with a passivating agent,e.g. a non-amino group containing silane, in order to reduce the aminogroup density on the surface, if necessary. The amino group-containingreactant may be diluted to a concentration of e.g. 1-50% with apassivating agent, preferably with the reaction product between glycidyloxypropyl trimethoxy silane and 3-mercapto 1,2-propanediol.Alternatively, the amino group density may be increased by using di- orpolyfunctional agents, which contain 2 or more primary or secondaryamino groups such as 1,13-diamino-4,7,10-trioxatridexane. Thereby, theaffinity of the matrix for His-tagged proteins may be increased. Thus,the reaction conditions may be chosen to obtain a final product withdesired characteristics, e.g. such that His-tagged proteins bindspecifically to the final product, while background-binding of otherproteins is minimal. The optimal amino group density may be determinedempirically for each solid support according to procedures described inthe Examples.

For example, the purification of proteins with a long histidine tag,with e.g. ≧10 histidine residues, is preferably carried out with asurface having a lower density of Ni²⁺-complexed polycarboxylic acidamide groups than purification of proteins having a tag of 6 histidineresidues. Conversely, purification under denaturing conditions, e.g. inthe presence of guanidinium chloride, is preferably carried out with ahigher density of chelating groups than a purification under nativeconditions.

In the next step, a condensation reaction between one carboxyl group ofthe polycarboxylic acid and an amino group on the surface is carriedout. FIG. 1 shows a schematic depiction of this reaction exemplifiedwith EDTA as polycarboxylic acid.

The reaction is carried out in the presence of a condensing agent, whichmay be selected from carbodiimides, carbonates such asdi(N-succinimidyl)carbonate,O—(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate oranalogous compounds. Carbodiimides such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), orN,N′-dicyclohexylcarbodiimide (DCC) are preferred. Reaction conditionswere found, where a single carboxyl group of the polycarboxylic acidreacts with the amino groups on the solid phase when the polycarboxylicacid is present in a sufficient molar excess over the amino groupsand/or the condensing agent. In preferred embodiment, to obtain a highlyspecific matrix for IMAC, reaction conditions are selected wherein theamino groups are reacted substantially quantitatively with thepolycarboxylic acid. Therefore, an at least 2-fold molar excess,preferably an at least 4-fold molar excess, of the condensing agent andan at least 5-fold excess, preferably an at least 10-fold molar excessand more preferably an at least 25-fold molar excess, of thepolycarboxylic acid over amino groups should be used (see Examples).

The reaction is preferably carried out in an aqueous phase. The reactionconditions are preferably pH 5-9, more preferably pH 7-8.5. Thepolycarboxylic acid is preferably used close to the saturation limit(e.g. 0.5 M for EDTA and 0.1 M for EGTA) and the condensing agent at1/10- 1/50 of the concentration of the polycarboxylic acid.

The reaction product is a solid phase having a polycarboxylic acidimmobilized thereto via a stable carboxylic acid amide bond. Preferably,the solid phase has the structure of immobilized formula (Ia) asindicated above. Preferably, the immobilized polycarboxylic acid residuehas still at least 3, 4 or more carboxyl groups and optionally furtherchelating groups such as amino or ether groups.

If the polycarboxylic acid is EDTA the solid phase preferably has astructure of formula (II):

wherein SP is the solid phase.

The solid phase of the invention, e.g. the EDTA-amide solid phase has ahigh affinity for polyvalent metal ions, such as transition metal ions,e.g. Ni²⁺, Zn²⁺, Co²⁺, or Cu²⁺-ions. Several combinations of these metalions with chelators immobilized according to this invention providehighly selective matrices for binding histidine-tagged proteins, withless non-specific binding than the traditionally immobilised NTA-group(see FIGS. 2 and 3).

In a preferred embodiment, the solid phase of the invention issubstantially quantitatively free from accessible amino groups, e.g.primary amino groups, and/or hydroxy groups, since unreacted aminogroups may be cause of low affinity Ni²⁺ binding and high unspecificbackground binding of polypeptides not carrying a His-tag. Thus, it maybe desirable that ≧90%, preferably ≧95% and more preferably ≧99% ofaccessible amino groups, particularly primary amino groups on thesurface of the solid phase are blocked, e.g. by reaction with thepolycarboxylic acid mediated by the condensing agent. The quantity ofaccessible unreacted groups, e.g. primary amino groups, may bedetermined according to known methods, e.g. by a ninhydrin-test or anOPA (ortho-phthalaldehyde/mercaptane) reaction.

Further, it is preferred that the solid phase, e.g. the EDTA-amide solidphase, is substantially free from loosely bound polyvalent metal ions,such as Ni²⁺ ions. The present inventors have found that when initiallysaturating an EDTA-amide matrix with Ni²⁺ ions, a significant amountthereof may remain only loosely bound thereto, in particular, when thesolid support still contains unreacted amine groups. This loosely boundNi²⁺ fraction may be detected with suitable reagents, e.g.dimethylglyoxime. For example, after adding 0.2-1 volumes of 1% w/vdimethylglyoxime dissolved in ethanol to an Ni²⁺ loaded matrix inTris-buffer pH7.5 and shaking the mixture for 15 minutes at 60° C., anyloosely bound Ni²⁺ ions can be detected as a pink precipitate. Looselybound metal ions may cause problems, namely a contamination of aprotein-containing sample with toxic and oxidizing Ni²⁺ ions and anincreased non-specific binding of non-His-tag proteins.

Thus, the solid phase of the present invention is preferablysubstantially free from loosely bound metal ions, particularly looselybound Ni²⁺ ions. Loosely bound metal ions may, e.g. be removed from thesolid phase by contacting the solid phase with a free polycarboxylicacid chelator, e.g. NTA or EGTA or EDTA, preferably at about pH 7.5,e.g. with 40 mM NTA or 10 mM EGTA or 10 mM EDTA, until loosely boundmetal ions have become undetectable, e.g. by contacting the matrix witha suitable detection reagent for the respective metal ion, e.g.dimethylglyoxime for the detection of Ni²⁺ ions. After removal ofloosely bound metal ions, the remaining ions remain so tightly boundthat even an overnight incubation at room temperature with an equalvolume of 0.5 M EDTA (pH 7.5) is insufficient for a complete releasefrom the EDTA-amide matrix. In contrast, it should be noted thatpre-washing of commercially available Ni-NTA-agarose (Qiagen) or of anNi-NTA-amide matrix with 40 mM NTA, or 10 mM EGTA or 10 mM EDTA removesnot only loosely bound, but apparently all bound Ni²⁺ ions (FIG. 4).

Surprisingly, no indication was found that the conversion of onecarboxyl group in EDTA to a carboxamide group would weaken the affinityfor Ni²⁺ at neutral pH. Instead, the carbonyl oxygen or the amino groupof the carboxamide can apparently engage in a coordinative bond to Ni²⁺,suggesting that EDTA-amide is a truly hexadentate chelator. That acarboxamide groups can coordinate Ni²⁺ is evident, e.g., from methylcoenzyme M-reductases, where 4 coordination sites for Ni²⁺ are providedby Coenzyme F430 and the 5^(th) by the carboxamide of a glutamine sidechain (Ermler et al, Science 278 (1997), 1457).

The solid phase of the invention may be used for immobilized metal ionchromatography (IMAC). In this chromatographic method polypeptidescomprising a plurality of histidine residues, e.g. consecutive histidineresidues, e.g. at least six consecutive histidine residues areselectively bound to a metal complex solid phase of the presentinvention and separated from other components. The polypeptide may beeluted from the solid phase by adding a suitable elution agent such asimidazole. The imidazole concentration is preferably in the range from 1to 1000 mM, depending on the density of immobilised active groups, thelength of the poly-histidine tag, and the multimeric state of the taggedprotein.

For IMAC purification of His-tagged proteins to work, it was so farassumed that the immobilised chelator should occupy only 3-5 out of the6 coordination sites of Ni²⁺. It is therefore an unexpected aspect ofthis invention that immobilised hexadentate chelators such as EDTA-amideor EDTA-hydroxamide and even chelators with more than 6 coordinatinggroups work extremely well for this application. Apparently, histidineside chains can transiently displace weakly coordinating groups within agiven chelator. Thus, according to an embodiment of the invention, theimmobilized chelator, i.e. the immobilized polycarboxylic acid amide has6 or more coordination groups, particularly selected from amino,carboxyl, carboxamide, and hydroxamate groups.

On the solid phase of the invention, e.g. EDTA-amide, kinetically verystable complexes e.g. with Ni²⁺ may be formed. The resulting matricesare extremely resistant to Ni²⁺-extraction, e.g. by EGTA or NTA. Even0.5 M EDTA extracts Ni²⁺ only very slowly, i.e. at a time scale of hoursto days. The resistance towards Ni²⁺-extraction increases further byincluding imidazole, e.g. 1 mM imidazole in the buffer. Therefore, IMACwith a solid phase of the invention, e.g. an EDTA-amide matrix, can beperformed in the presence of a chelating metalloprotease-inhibitor, e.g.EDTA in concentrations of 1-10 mM, e.g. 5 mM.

Traditional Ni-chelate matrices, such as Ni-NTA agarose, get easilyreduced by protein-protecting agents such as dithiothreitol (DTT). Evenlow millimolar concentrations of DTT extract nickel in the form ofbrownish reduction products. In contrast, Ni²⁺ EDTA-amide matrices, fromwhich loosely-bound Ni²⁺ had been removed, do not show these problems.Instead, on Ni²⁺ EDTA-amide silica we could successfully purifyhis-tagged proteins in the presence of extremely high concentrations ofDTT, e.g. of 0.5 M or 1 M DTT (FIG. 5), which is a 100-1000-fold higherDTT concentration than used in typical protein purification schemes.Thus, in a preferred embodiment, the metal ion, e.g. Ni²⁺ ion containingsolid phase of the invention is stable in the presence of athiol-containing or dithiol-containing reducing agent, e.g., DTT, in aconcentration of at least 10 mM, preferably of at least 20 mM, morepreferably of at least 50 mM, and even more preferably of at least 100mM or even of at least 500 mM, and up to 1000 mM or even higher,preferably for at least 1 h at room temperature.

In an especially preferred embodiment of the present invention thechromatography is carried out with a recombinant polypeptide comprisinga plurality of histidine residues in a “spaced histidine tag”,preferably in a sequence [H_(n)S_(m)]_(k) wherein H is histidine, S aspacer residue selected from glycine and/or serine and/or threonine, nis 1-4, m is 1-6, preferably 1-4, and k is 2-8, preferably 2-6. Thelength of the tag is preferably from 8 to 50 amino acids, morepreferably from 12 to 40 amino acids. The present invention alsocomprises irregularily-spaced histidine tags, where the lengths of theoligo histidine clusters and the lengths of the spacer regions varywithin a given tag sequence. The spaced histidine tag is preferablylocated at the N- and/or at the C-terminus of the recombinantpolypeptide and/or inserted into the sequence of the recombinantpolypeptide.

Further, the invention is explained in more detail by the followingFigures and Examples.

FIGURE LEGENDS

FIG. 1: Scheme for the coupling of EDTA to an amine-containing support.The reaction is carried out under conditions where a selective reactionoccurs between a single carboxyl group of EDTA with the amino group onthe support.

FIG. 2: Comparison of the protein binding specificity of variousNi²⁺-containing chromatographic matrices in IMAC.

The protein binding characteristics of various His-tagged proteins,namely a comparative matrix (NTA-Qiagen) and three inventive matrices(EDTA-amide silica I, EDTA-amide silica II and EDTA-amide sepharose) areshown (cf. Example 5 for details).

FIG. 3: Combination of various chelators and transition metal ions forIMAC.

The protein binding properties of various matrices (inventive EDTA, EGTAand TTHA matrices and an NTA matrix) with different chelators in thepresence of various transition metal ions are shown (cf. Example 6 fordetails).

FIG. 4: Resistance of Ni-chelate matrices against extraction of Ni²⁺ byfree chelators.

The strength of Ni²⁺ binding to an inventive EDTA-amide matrix andcomparative NTA matrices is shown (cf. Example 7 for details).

FIG. 5: Resistance of various Ni-chelate matrices againstthiol-containing reducing agents.

The resistance of various Ni²⁺ containing chelator matrices (aninventive EDTA-amide matrix and comparative NTA matrices) against Ni²⁺extraction and reduction in the presence of DTT is shown (cf. Example 8for details).

FIG. 6: Characterisation of spaced poly-histidine tags.

A) The binding characteristics of inventive spaced poly-His tags (spacedH14, spaced H21 and spaced H28) and comparative His tags (MRGS6 and H10)are shown.

B) The expression of proteins containing an inventive spaced His-tag anda comparative His-tag (H10) are shown (cf. Example 9 for details).

EXAMPLES Example 1 Preparation of Ni-EDTA-Amide Sepharose 4B

1 liter Sepharose 4B is prewashed on a glass-funnel with 1 liter 0.1 MNaOH (in water), followed by 5 times one liter of pure water,transferred to a 5 liter flask, and filled up with water to a volume of2 liters. 0.8 mol of NaOH are added, the temperature adjusted to 25° C.,followed by addition of 1.0 mol epibromhydrine. The mixture is thenshaken for 2 hours at 25° C., and then chilled on ice. The resultingepoxy-activated Sepharose is subsequently recovered by filtrationthrough a glass funnel, washed with water, and resuspended in 2 M NH₄Cl(final concentration in water, final volume 2 liters). 4 mol NH₃ areadded from a 25% aqueous solution and the mixture is shaken o/n at roomtemperature.

The resulting NH₂-Sepharose 4B is recovered by filtration, washed withwater until free NH₃ has be become undetectable, and resuspended in 0.5M EDTA/Na⁺ pH 8.0 (final concentration, final volume 2 liters). Then, 50mmol EDC are added and the mixture is shaken for 1 hour at roomtemperature. Thereafter, another 50 mmol EDC aliquot is added and thereaction is allowed to proceed o/n.

The resulting EDTA-amide Sepharose is recovered by filtration and washeduntil the free EDTA-concentration has dropped below 1 mM. The Sepharoseis then charged with 20 mM NiCl₂ in Tris buffer pH 7.5, until free Ni²⁺appears in the non-bound fraction. Free and loosely-bound Ni²⁺ ions arethen removed by washing with water, 10 mM NTA pH 7.5, water, and isfinally resuspended in 30% ethanol+10 mM imidazole/HCl+1 mM NTA pH 7.5for long-term storage.

The properties of the Ni²⁺ EDTA-amide Sepharose can be adjusted byvarying the epoxy-activation step. Coupling at higher temperature (up to40° C.) and using higher concentrations of epichlorhydrine and NaOH (upto 1.2 M epichlorhydrine and 1.0 M NaOH, respectively) will result inhigher coupling-density, but also in higher background-binding. Lowertemperature (down to 18° C.) using a lower concentration ofepichlorhydrine and NaOH (down to 0.2 M epichlorhydrine and 0.1 M NaOH,respectively) will result in lower coupling density, and in even lowerbackground binding, but also in lower specific binding capacity, inparticular for proteins with short His-tags.

Example 2 Preparation of EDTA-Amide Magnetic Beads

5 ml of amine-terminated magnetic beads (Sigma #17643-5 ml) are washedin water and resuspended in a final volume of 5 ml in 0.5 M EDTA/Na⁺ pH8.0. A 125 μmol EDC aliquot is added and the reaction is shaken for 1hour at room temperature. Thereafter, another 125 μmol EDC aliquot isadded and the reaction is continued o/n.

The resulting EDTA-amide magnetic beads are recovered by magneticseparation, washed with water, and charged with Ni²⁺ or another metalion analogously to Example 1.

Example 3 Preparation of High-Density EDTA-Amide Silica

100 g Davisil XWP1000 Å 90-130 (Grace) are resuspended in 500 ml of 3%(v/v) aminopropyl triethoxy silane, 4% water, 93% ethanol and shakengently for 2 hours at 40° C. and then o/n at room temperature. Theamino-modified silica is recovered by filtration and free silane isremoved by washing with pure water. Covalent coupling to EDTA andcharging with Ni²⁺ are performed as described for Sepharose 4B. For longterm-storage, the product can be dried out of water or isopropanol.

Example 4 Preparation of EDTA-Amide Silica with a Passivated Surface

The Ni²⁺ EDTA-amide silica from Example 3 three still suffers from highbackground-binding when used in IMAC. This background may probablyresult from residual silanol- and amino groups (which for stericalreasons could not react with the activated EDTA) and/or from highsurface concentrations of EDTA-amide. The background-problem can besolved by including a passivating silane during the modification ofsilica with aminopropyl-silane. The so far best passivating silane isthe reaction product between glycidyl oxypropyl trimethoxy silane and3-mercapto 1,2-propandiol.

3 ml glycidyl oxypropyl trimethoxy silane are mixed with 3 ml 2-mercapto1,3-propandiol, 90 ml methanol, 4 ml water and 10 μl 4-methylmorpholine, and the reaction is allowed to proceed for 30 minutes at 25°C. 150 μl aminopropyl trimethoxy silane are added, and the resultingmixture is used to modify 30 g Davisil XWP1000 Å 90-130 as describedabove. The ratio between the aminosilane and the passivating silane isin this example defined as 5%. It can, however, be varied between 1% and50%. The resulting passivated amino-silica is then reacted with EDTA oranother chelating group, charged with metal ions, and treated to removeloosely-bound metal ions as described above.

Example 5 Comparison of Ni²⁺-Containing Chromatographic Supports in IMAC

The following chromatographic supports were tested:

“NTA-Qiagen”: Ni²⁺-NTA Agarose purchased from Qiagen “EDTA-amide silicaI”: Ni²⁺-charged EDTA-amide silica with 20% coupling density (Example 4)“EDTA-amide silica II”: Ni²⁺-charged EDTA-amide silica with 5% couplingdensity (Example 4) “EDTA-amide Sepharose ”: Ni²⁺-charged EDTA-amideSepharose 4B (Example 1)

E. coli cells were resuspended in buffer (50 mM Tris/HCl pH 7.5, 2 mMmagnesium acetate, 50 mM NaCl, 5 mM mercaptoethanol). A lysate wasprepared and cleared by ultracentrifugation. Then, either 1 μM of afusion protein (“NES-YFP-H₁₀”) comprising a nuclear export signal (NES),the yellow fluorescent protein (YFP) and a C-terminal deca His-tag, or 1μM of a fusion between the maltose binding protein (MBP) with aC-terminal hexa His-tag (“MBP-H₆”), or 0.5 μM of a fusion proteincontaining a deca His-tag, a zz-tag and the mouse exportin CRM1(“H₁₀-zz-CRM1”) were added and used as starting materials for thebinding experiments. For purification of the deca-His-tagged proteins,starting materials were supplemented with 1 mM imidazole.

400 μl starting material each were rotated o/n at 4° C. with 10 μlchromatographic support. After washing with 5 ml buffer, bound proteinswere eluted with 0.4 M imidazole pH 7.5. Analysis was by SDS-PAGE on 12%acrylamide gel, followed by Coomassie-staining. The load corresponds to0.5 μl matrix.

FIG. 2 shows the results for NES-YFP-H₁₀ (panel A), MBP-H₆ (panel B) andH₁₀-ZZ-CRM1 (panel C). All tested matrices exhibit efficient binding ofHis-tagged proteins. The matrices of the present invention however showsignificantly lower backgrounds compared to the NTA-Qiagen matrix.

Example 6 Testing of Various Chelators and Transition Metal Ions forIMAC

Amino-silica with 5% coupling density (as described in Example 4) wasconjugated with either EDTA, EGTA, NTA, or TTHA. Each of the resultingmatrices was then charged with either Ni²⁺, Co²⁺, Zn²⁺, or Cu²⁺. Thebinding assays for NES-YFP-H₁₀ were performed as described in Example 5,however, 100 mM NaCl were included in the wash buffer.

FIG. 3 shows that the tested combinations of matrices and metal ionsshow efficient binding of NES-YFP-H₁₀.

Example 7 Resistance of Ni-Chelate Matrices Against Extraction of Ni²⁺by Free Chelators

Each 1 ml of Ni²⁺-EDTA-amide silica (50% coupling density; preparedaccording to Example 4), Ni²⁺-NTA-amide silica (50% coupling density),or Ni²⁺-NTA-agarose (Qiagen) were washed over a time of 45 min witheither 30 ml Tris-buffer, or 10 mM EDTA pH 7.6, or 10 mM EGTA pH 7.6, or40 mM NTA pH 7.6, followed by equilibration in binding buffer (50 mMTris/HCl pH 7.5, 500 mM NaCl, 5 mM MgCl₂). 10 μl of each pre-treatedmatrix was then used to bind a His₁₀-MBP-GFP fusion (10 μMconcentration) out of 600 μl E. coli lysate. The bound fraction waseluted with 75 μl 1 M imidazol/HCl pH 7.5. 1 μl of each eluate was thenanalysed by SDS-PAGE followed by Coomassie-staining. The results areshown in FIG. 4A. The inventive Ni²⁺-EDTA-amide silica matrix wascompletely resistant against all tested chelator-solutions. Thecomparative matrices were significantly less resistant. Ni²⁺-NTA-agarosewas discharged by any of the chelator-treatments. Ni²⁺-NTA-amide silicawas completely discharged by 10 mM EDTA and 40 mM NTA, but retained asmall activity after treatment with EGTA.

The chelator-washed matrices from the panel shown in FIG. 4A wereequilibrated in 100 mM Tris/HCl pH 7.5, mixed with an equal volume of 1%dimethylglyoxime (dissolved in Ethanol) and incubated for 15 min at 60°and subsequently o/n at room temperature, before photographs were taken.The results are shown in FIG. 4B. A pink dimethylglyoxime-Ni²⁺precipitate, representing loosely-bound Ni²⁺, occurred with untreatedNi²⁺-NTA-amide silica and with untreated Ni²⁺-NTA agarose, but not withthe inventive Ni²⁺-EDTA-amide silica. Pre-washing of Ni²⁺-NTA-amidesilica or of Ni²⁺-NTA agarose with the above-mentioned chelators removedloosely-bound Ni²⁺ to the same extent as it reduced the capacity to bindthe histidine-tagged protein.

Thus, the binding of Ni²⁺ ions to the inventive EDTA-amide matrix issignificantly stronger than to comparative NTA matrices.

Example 8 Resistance of Ni-Chelator Matrices Against Thiol-ContainingReducing Agents

The resistance of various Ni²⁺ containing chelator matrices againstdithiothreitol (DTT), a thiol-containing reducing agent, was tested.

NTA-agarose (Qiagen), NTA-amide silica and EDTA-amide silica were eitherleft untreated or were incubated o/n at room temperature with 1 M DTT,buffered with 1 M Tris/HCl pH 7.5. The results are shown in FIG. 5A. TheDTT treatment converted Ni²⁺ from NTA-agarose (Qiagen) and NTA-amidesilica into brownish reduction products. In contrast, Ni²⁺ EDTA-amidesilica remained fully unaffected.

The above matrices (≈50 μl) were resuspended in either 1 ml 1 M Tris/HClpH 7.5 or 1 M Tris/HCl pH 7.5+1 M DTT. 5 min later, a His₁₀-tagged redfluorescent protein was added and the binding reactions were rotated o/nat room temperature. The matrices were allowed to sediment by gravityand photographs were taken. The results are shown in FIG. 5B (upperpanel).

Red colour on the beads indicates binding of the his-tagged protein. Inthe absence of DTT, all matrices bound the his-tagged protein very well.The DTT-treatment fully abolished binding to Ni²⁺ NTA agarose (Qiagen)and Ni²⁺ NTA-amide silica. The non-bound fractions contained brownishreduction products of Ni²⁺ that were released from the beads. Incontrast, binding of the His-tagged red fluorescent protein to theinventive Ni²⁺ EDTA amide silica remained unaffected by the DTTtreatment.

The bound fractions were eluted with 1 M imidazol pH 7.3, and analysedby SDS-PAGE followed by Coomassie staining. The results are shown inFIG. 5B (lower panel). The analysis confirmed that 1 M DTT completelyabolished binding of the his-tagged protein to Ni²⁺ NTA-agarose or Ni²⁺NTA-amide silica, while binding to the inventive Ni²⁺ EDTA-amide silicawas not affected at all.

Example 9 Characterisation of Spaced Poly-Histidine Tags

A mixture of DHFR derivatives tagged with various histidine tags wasbound to Ni²⁺ EDTA amide silica and eluted slowly with a gradient ofincreasing imidazole concentration. The results are shown in FIG. 6A.(Actual concentrations are given above the lanes). Panel shows analysisof eluted fractions by SDS-PAGE/Coomassie-staining. Tags with greaternumber of histidines confer tighter binding to the matrix, elution athigher imidazole concentration, and thus better separation fromcontaminants that are not poly-histidine tagged. The following tags havebeen used (amino acids in single letter code):

(SEQ ID NO: 1) MRGS6 = MRGSHHHHHH (SEQ ID NO: 2) H10 = MHHHHHHHHHH(SEQ ID NO: 3) spaced H14 = MSKHHHHSGHHHTGHHHHSGSHHH (SEQ ID NO: 4)spaced H21 = MSKHHHHSGHHHTGHHHHSGSHHHTGHHHHSGSHHH (SEQ ID NO: 5)spaced H28 = MSKHHHHSGHHHTGHHHHSGSHHHTGHHHHSGSHHH TGHHHHSGSHHH

Although His-tags with more histidines confer a more specific binding toNi-chelate matrices, they pose the problem that continuous stretches oftoo many histidines compromises expression levels and solubility in E.coli. Interrupting continuous polyhistidine stretches with Gly, Ser,and/or Thre-containing spacers rectifies these problems. In therepresentative example shown, tagging of DHFR with a spaced His₁₄ tagdoubled the yield of soluble protein during recombinant expression in E.coli as compared to a conventional, continuous His₁₀ tag (FIG. 6B).

1-21. (canceled)
 22. A method for immobilized metal ion chromatography(IMAC), comprising contacting a sample with an immobilized chelatorhaving 6 or more coordination groups.
 23. The method of claim 22,wherein the immobilized chelator is a polycarboxylic acid amide or esterhaving 6 or more coordination groups.
 24. The method according to claim23, wherein said coordination groups are selected from the groupconsisting of amino, carboxyl, carboxamide and hydroxamate groups. 25.The method of claim 22, wherein the immobilized chelator is a solidphase having a polycarboxylic acid immobilized thereto, wherein saidimmobilized chelator has a structure of formula (Ia) or (Ib):

wherein SP is a solid phase; R¹ is hydrogen or an organic residue thatdoes not interfere with the application of the solid phase, and PCA isthe residue of a polycarboxylic acid.
 26. The method of claim 25,wherein said PCA is the residue of an amino polycarboxylic acid or asalt thereof.
 27. The method of claim 25, wherein the polycarboxylicacid is selected from the group consisting of ethylene diaminotetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),diethylene triamino pentaacetic acid (DPTA), and triethylenetetramine-N,N,N′,N′, N″,N″-hexa-acetic acid (TTHA), salts thereof. 28.The method according to claim 27, wherein said polycarboxylic acid isethylene diamino tetraacetic acid (EDTA).
 29. The method of claim 25,wherein the solid phase has a structure of formula (II):

wherein SP is the solid phase, and wherein one or more of the carboxylicacid groups may be deprotonated.
 30. The method of claim 22, wherein thechelator is complexed with a metal ion.
 31. The method according toclaim 30, wherein said metal ion is a transition metal ion.
 32. Themethod according to claim 31, wherein said transition metal a Ni²⁺ ion.33. A method for binding a polycarboxylic acid having 6 or morecoordination groups to a solid phase, comprising the steps: (a)providing a polycarboxylic acid and a solid phase comprising aminogroups, (b) reacting the amino groups with the polycarboxylic acid inthe presence of a condensing agent, wherein the condensing agent ispresent in a molar excess over the amino groups and the polycarboxylicacid is present in a molar excess over the condensing agent and aminogroups, and wherein a single carboxyl group of the polycarboxylic acidreacts with the amino groups.
 34. The method of claim 33, wherein thepolycarboxylic acid is an amino polycarboxylic acid selected from thegroup consisting of ethylene diamino tetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),diethylene triamino pentaacetic acid (DPTA), and triethylenetetramine-N,N,N′,N′, N″,N″-hexa-acetic acid (TTHA).
 35. The method ofclaim 33, wherein the solid phase is an amino-functionalizedchromatographic support, particularly selected from amino-functionalizedcarbohydrates such as agarose, sepharose, or cellulose, metals orsemi-metals such as silicon, or metal oxides or semi-metal oxides suchas silica, glass, plastics such as polystyrene or lipids such asphospholipids.
 36. The method of claim 33, wherein the solid phasecomprises particles, e.g. vesicles, magnetic beads, quantum dots,proteins, or molecules with multiple primary or secondary amino groups.37. The method of claim 33, wherein the amino groups on the solid phaseare primary or secondary amino groups, e.g. aliphatic primary aminogroups.
 38. The method of claim 33, wherein the condensing agent isselected from carbodiimides such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), orN,N′-dicyclohexylcarbodiimide (DCC).
 39. The method of claim 33, whereinthe molar excess of the condensing agent over the amino groups is atleast 2-fold, preferably at least 4-fold, and/or wherein the molarexcess of the polycarboxylic acid over the amino groups is at least5-fold, preferably at least 25-fold.
 40. A solid phase having apolycarboxylic acid immobilized thereto having a structure of formula(Ia) or (Ib):

wherein SP is a solid phase; R¹ is hydrogen or an organic residue thatdoes not interfere with the application of a solid phase, and PCA is theresidue of a polycarboxylic acid, particularly of an aminopolycarboxylic acid, or a salt thereof, wherein the immobilizedpolycarboxylic acid amide or ester has at least 6 or more coordinationgroups, which are particularly selected from amino, carboxyl,carboxamide and hydroxamate groups, and wherein the surface of the solidphase is preferably substantially free from accessible amino and/orhydroxy groups, wherein e.g. ≧90%, preferably ≧95% and more preferably≧99% of the accessible amino and/or hydroxy groups on the surface of thesolid phase are blocked.
 41. The solid phase of claim 40, wherein PCA isthe residue of EDTA or a salt thereof.
 42. The solid phase of claim 40having a structure of formula (II):

wherein SP is the solid phase and one or more of the carboxylic acidgroups may be deprotonated.
 43. The solid phase of claim 40, wherein thepolycarboxylic residue is complexed with a metal ion, e.g. a transitionmetal ion, such as a Ni²⁺ ion, and wherein substantially all looselybound metal ions preferably have been removed.
 44. The solid phase ofclaim 43, which is stable in the presence of a thiol- ordithiol-containing reducing agent.
 45. A method of purifying arecombinant polypeptide comprising the steps: (a) providing a samplecomprising a polypeptide with a plurality of histidine residues, (b)contacting the sample with a solid phase of claim 39 that contains apre-bound complexing metal ion, e.g. a Ni²⁺ ion, under conditionswherein the recombinant polypeptide selectively binds to the solidphase, (c) separating the bound recombinant polypeptide from othersample components, and (d) eluting the recombinant polypeptide from thesolid phase.
 46. The method of claim 45, wherein the polypeptidecomprises at least 6 consecutive histidine residues.
 47. The method ofclaim 45, wherein the polypeptide comprises at least 4 histidineresidues in a polyhistidine sequence [H_(n)S_(m)]_(k) wherein H is ahistidine residue and S is a spacer amino acid residue, selected fromglycine and/or serine and/or threonine, n is in each case independently1-4, m is in each case independently 1-6, and k is 2-6.
 48. Arecombinant polypeptide comprising at least 4 histidine residues in asequence [H_(n)S_(m)]_(k) wherein H is a histidine residue and S is aspacer amino acid residue, selected from glycine and/or serine and/orthreonine, n is in each case independently 1-4, m is in each caseindependently 1-6, and k is 2-8.
 49. The polypeptide of claim 48,wherein the polyhistidine sequence is at the N- and/or at theC-terminus, or inserted into the sequence of the tagged protein.